Investigating the human and nonobese diabetic mouse MHC class II immunopeptidome using protein language modeling.

MOTIVATION: Identifying peptides associated with the major histocompability complex class II (MHCII) is a central task in the evaluation of the immunoregulatory function of therapeutics and drug prototypes. MHCII-peptide presentation prediction has multiple biopharmaceutical applications, including the safety assessment of biologics and engineered derivatives in silico, or the fast progression of antigen-specific immunomodulatory drug discovery programs in immune disease and cancer. This has resulted in the collection of large-scale datasets on adaptive immune receptor antigenic responses and MHC-associated peptide proteomics. In parallel, recent deep learning algorithmic advances in protein language modeling have shown potential in leveraging large collections of sequence data and improve MHC presentation prediction. RESULTS: Here, we train a compact transformer model (AEGIS) on human and mouse MHCII immunopeptidome data, including a preclinical murine model, and evaluate its performance on the peptide presentation prediction task. We show that the transformer performs on par with existing deep learning algorithms and that combining datasets from multiple organisms increases model performance. We trained variants of the model with and without MHCII information. In both alternatives, the inclusion of peptides presented by the I-Ag7 MHC class II molecule expressed by nonobese diabetic mice enabled for the first time the accurate in silico prediction of presented peptides in a preclinical type 1 diabetes model organism, which has promising therapeutic applications. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/Novartis/AEGIS.

A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab.

Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.

Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation.

Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and in silico metagenomic approaches, we retrieved 16 esterases from the α/ß-hydrolase fold superfamily with the closest match from uncultured acidophilic Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium, and Ferrimicrobium), Acidiphilium, and other Proteobacteria inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in Escherichia coli. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33-68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30-65°C, retaining 20-61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U/g at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.

Plasma and stool metabolomics to identify microbiota derived-biomarkers of metabolic dysfunction-associated fatty liver disease: effect of PNPLA3 genotype.

INTRODUCTION: Non-invasive biomarkers are needed for metabolic dysfunction-associated fatty liver disease (MAFLD), especially for patients at risk of disease progression in high-prevalence areas. The microbiota and its metabolites represent a niche for MAFLD biomarker discovery. However, studies are not reproducible as the microbiota is variable. OBJECTIVES: We aimed to identify microbiota-derived metabolomic biomarkers that may contribute to the higher MAFLD prevalence and different disease severity in Latin America, where data is scarce. METHODS: We compared the plasma and stool metabolomes, gene patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 single nucleotide polymorphism (SNP), diet, demographic and clinical data of 33 patients (12 simple steatosis and 21 steatohepatitis) and 19 healthy volunteers (HV). The potential predictive utility of the identified biomarkers for MAFLD diagnosis and progression was evaluated by logistic regression modelling and ROC curves. RESULTS: Twenty-four (22 in plasma and 2 in stool) out of 424 metabolites differed among groups. Plasma triglyceride (TG) levels were higher among MAFLD patients, whereas plasma phosphatidylcholine (PC) and lysoPC levels were lower among HV. The PNPLA3 risk genotype was related to higher plasma levels of eicosenoic acid or fatty acid 20:1 (FA(20:1)). Body mass index and plasma levels of PCaaC24:0, FA(20:1) and TG (16:1_34:1) showed the best AUROC for MAFLD diagnosis, whereas steatosis and steatohepatitis could be discriminated with plasma levels of PCaaC24:0 and PCaeC40:1. CONCLUSION: This study identified for the first time MAFLD potential non-invasive biomarkers in a Latin American population. The association of PNPLA3 genotype with FA(20:1) suggests a novel metabolic pathway influencing MAFLD pathogenesis.

A body weight loss- and health-promoting gut microbiota is established after bariatric surgery in individuals with severe obesity.

Obesity has reached an epidemic level worldwide, and bariatric surgery (BS) has been proven to be the most efficient therapy to reduce severe obesity-related comorbidities. Given that the gut microbiota plays a causal role in obesity development and that surgery may alter the gut environment, investigating the impact of BS on the microbiota in the context of severe obesity is important. Although, alterations at the level of total gut bacteria, total gene content and total metabolite content have started to be disentangled, a clear deficit exists regarding the analysis of the active fraction of the microbiota, which is the fraction that is most reactive to the BS. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics and metabolomics in 40 severely obese volunteers. Samples from each volunteer were obtained under basal conditions, after a short high protein and calorie-restricted diet, and 1 and 3 months after BS, including laparoscopic surgery through Roux-en-Y Gastric Bypass or Sleeve Gastrectomy. The results revealed for the first time the most active microbes and metabolic flux distribution pre- and post-surgery and deciphered main differences in the way sugars and short-fatty acids are metabolized, demonstrating that less energy-generating and anaerobic metabolism and detoxification mechanisms are promoted post-surgery. A comparison with non-obese proteome data further signified different ways to metabolize sugars and produce short chain fatty acids and deficiencies in proteins involved in iron transport and metabolism in severely obese individuals compared to lean individuals.

Functional microbiome deficits associated with ageing: Chronological age threshold.

Composition of the gut microbiota changes during ageing, but questions remain about whether age is also associated with deficits in microbiome function and whether these changes occur sharply or progressively. The ability to define these deficits in populations of different ages may help determine a chronological age threshold at which deficits occur and subsequently identify innovative dietary strategies for active and healthy ageing. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics in three well-defined age groups consisting of 30 healthy volunteers, namely, ten infants, ten adults and ten elderly individuals. Samples from each volunteer at intervals of up to 6 months (n = 83 samples) were used for validation. Ageing gradually increases the diversity of gut bacteria that actively synthesize proteins, that is by 1.4-fold from infants to elderly individuals. An analysis of functional deficits consistently identifies a relationship between tryptophan and indole metabolism and ageing (p < 2.8e-8 ). Indeed, the synthesis of proteins involved in tryptophan and indole production and the faecal concentrations of these metabolites are directly correlated (r2  > .987) and progressively decrease with age (r2  > .948). An age threshold for a 50% decrease is observed ca. 11-31 years old, and a greater than 90% reduction is observed from the ages of 34-54 years. Based on recent investigations linking tryptophan with abundance of indole and other "healthy" longevity molecules and on the results from this small cohort study, dietary interventions aimed at manipulating tryptophan deficits since a relatively "young" age of 34 and, particularly, in the elderly are recommended.

Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7α-dehydroxylating anaerobes. While this organism is well-studied relative to bile acid metabolism, little is known about the basic nutrition and physiology of C. scindens ATCC 35704. To determine the amino acid and vitamin requirements of C. scindens, the leave-one-out (one amino acid group or vitamin) technique was used to eliminate the nonessential amino acids and vitamins. With this approach, the amino acid tryptophan and three vitamins (riboflavin, pantothenate, and pyridoxal) were found to be required for the growth of C. scindens In the newly developed defined medium, C. scindens fermented glucose mainly to ethanol, acetate, formate, and H2. The genome of C. scindens ATCC 35704 was completed through PacBio sequencing. Pathway analysis of the genome sequence coupled with transcriptome sequencing (RNA-Seq) under defined culture conditions revealed consistency with the growth requirements and end products of glucose metabolism. Induction with bile acids revealed complex and differential responses to cholic acid and deoxycholic acid, including the expression of potentially novel bile acid-inducible genes involved in cholic acid metabolism. Responses to toxic deoxycholic acid included expression of genes predicted to be involved in DNA repair, oxidative stress, cell wall maintenance/metabolism, chaperone synthesis, and downregulation of one-third of the genome. These analyses provide valuable insight into the overall biology of C. scindens which may be important in treatment of disease associated with increased colonic secondary bile acids.IMPORTANCEC. scindens is one of a few identified gut bacterial species capable of converting host cholic acid into disease-associated secondary bile acids such as deoxycholic acid. The current work represents an important advance in understanding the nutritional requirements and response to bile acids of the medically important human gut bacterium, C. scindens ATCC 35704. A defined medium has been developed which will further the understanding of bile acid metabolism in the context of growth substrates, cofactors, and other metabolites in the vertebrate gut. Analysis of the complete genome supports the nutritional requirements reported here. Genome-wide transcriptomic analysis of gene expression in the presence of cholic acid and deoxycholic acid provides a unique insight into the complex response of C. scindens ATCC 35704 to primary and secondary bile acids. Also revealed are genes with the potential to function in bile acid transport and metabolism.

Decoding the ocean's microbiological secrets for marine enzyme biodiscovery.

A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 × 1018 m3 and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 1012 cells per millilitre, exceeding eukaryotic densities of around 106 cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project 'INMARE'.

Identification of Halophilic Microbes in Lung Fibrotic Tissue by Oligotyping.

Idiopathic pulmonary fibrosis (IPF) is an incurable disease with poor prognosis and unknown etiology. The poor clinical outcome is associated with enhanced microbial burden in bronchoalveolar lavage fluid from IPF patients. However, whether microbes from the respiratory tract fluid cause the disease remains uncertain. Tissue-associated microbes can influence host physiology in health and disease development. The aim of this study was to evaluate the existence of microbes in lung fibrotic tissues. We evaluated the microbial community in lung tissues from IPF and from human transforming growth factor-ß1 (TGF-ß1) transgenic mice with lung fibrosis by oligotyping. We also evaluated the microbial population in non-tumor-bearing tissues from surgical specimens of lung cancer patients. The phyla Firmicutes and the genus Clostridium tended to be predominant in the lung tissue from IPF and lung cancer patients. Oligotyping analysis revealed a predominance of bacteria belonging to the genera Halomonas, Shewanella, Christensenella, and Clostridium in lung tissue from IPF and lung cancer. Evaluation of the microbial community in the lung tissue from mice revealed abundance of Proteobacteria in both wild-type (WT) littermates and transgenic mice. However, the genus Halomonas tended to be more abundant in TGF-ß1 transgenic mice compared to WT mice. In conclusion, this study describes tissue-associated microbes in lung fibrotic tissues from IPF patients and from aging TGF-ß1 transgenic mice.

Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243T.

Strains of Eggerthella lenta are capable of oxidation-reduction reactions capable of oxidizing and epimerizing bile acid hydroxyl groups. Several genes encoding these enzymes, known as hydroxysteroid dehydrogenases (HSDH) have yet to be identified. It is also uncertain whether the products of E. lenta bile acid metabolism are further metabolized by other members of the gut microbiota. We characterized a novel human fecal isolate identified as E. lenta strain C592. The complete genome of E. lenta strain C592 was sequenced and comparative genomics with the type strain (DSM 2243) revealed high conservation, but some notable differences. E. lenta strain C592 falls into group III, possessing 3α, 3ß, 7α, and 12α-hydroxysteroid dehydrogenase (HSDH) activity, as determined by mass spectrometry of thin layer chromatography (TLC) separated metabolites of primary and secondary bile acids. Incubation of E. lenta oxo-bile acid and iso-bile acid metabolites with whole-cells of the high-activity bile acid 7α-dehydroxylating bacterium, Clostridium scindens VPI 12708, resulted in minimal conversion of oxo-derivatives to lithocholic acid (LCA). Further, Iso-chenodeoxycholic acid (iso-CDCA; 3ß,7α-dihydroxy-5ß-cholan-24-oic acid) was not metabolized by C. scindens. We then located a gene encoding a novel 12α-HSDH in E. lenta DSM 2243, also encoded by strain C592, and the recombinant purified enzyme was characterized and substrate-specificity determined. Genomic analysis revealed genes encoding an Rnf complex (rnfABCDEG), an energy conserving hydrogenase (echABCDEF) complex, as well as what appears to be a complete Wood-Ljungdahl pathway. Our prediction that by changing the gas atmosphere from nitrogen to hydrogen, bile acid oxidation would be inhibited, was confirmed. These results suggest that E. lenta is an important bile acid metabolizing gut microbe and that the gas atmosphere may be an important and overlooked regulator of bile acid metabolism in the gut.

Targeted Synthesis and Characterization of a Gene Cluster Encoding NAD(P)H-Dependent 3α-, 3ß-, and 12α-Hydroxysteroid Dehydrogenases from Eggerthella CAG:298, a Gut Metagenomic Sequence.

Gut metagenomic sequences provide a rich source of microbial genes, the majority of which are annotated by homology or unknown. Genes and gene pathways that encode enzymes catalyzing biotransformation of host bile acids are important to identify in gut metagenomic sequences due to the importance of bile acids in gut microbiome structure and host physiology. Hydroxysteroid dehydrogenases (HSDHs) are pyridine nucleotide-dependent enzymes with stereospecificity and regiospecificity for bile acid and steroid hydroxyl groups. HSDHs have been identified in several protein families, including medium-chain and short-chain dehydrogenase/reductase families as well as the aldo-keto reductase family. These protein families are large and contain diverse functionalities, making prediction of HSDH-encoding genes difficult and necessitating biochemical characterization. We located a gene cluster in Eggerthella sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in Escherichia coli We then screened bile acid substrates against the purified recombinant enzymes. CDD59475 is a novel 12α-HSDH, and we determined that CDD59474 (3α-HSDH) and CDD59473 (3ß-HSDH) constitute novel enzymes in an iso-bile acid pathway. Phylogenetic analysis of these HSDHs with other gut bacterial HSDHs and closest homologues in the database revealed predictable clustering of HSDHs by function and identified several likely HSDH sequences from bacteria isolated or sequenced from diverse mammalian and avian gut samples.IMPORTANCE Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids, with implications for increased/decreased toxicity for gut bacteria and the host. The generation of oxo-bile acids is known to inhibit host enzymes involved in glucocorticoid metabolism and may alter signaling through nuclear receptors such as farnesoid X receptor and G-protein-coupled receptor TGR5. Biochemical or similar approaches are required to fill in many gaps in our ability to link a particular enzymatic function with a nucleic acid or amino acid sequence. In this regard, we have identified a novel 12α-HSDH and a novel set of genes encoding an iso-bile acid pathway (3α-HSDH and 3ß-HSDH) involved in epimerization and detoxification of harmful secondary bile acids.

Metabolic versatility of small archaea Micrarchaeota and Parvarchaeota.

Small acidophilic archaea belonging to Micrarchaeota and Parvarchaeota phyla are known to physically interact with some Thermoplasmatales members in nature. However, due to a lack of cultivation and limited genomes on hand, their biodiversity, metabolisms, and physiologies remain largely unresolved. Here, we obtained 39 genomes from acid mine drainage (AMD) and hot spring environments around the world. 16S rRNA gene based analyses revealed that Parvarchaeota were only detected in AMD and hot spring habitats, while Micrarchaeota were also detected in others including soil, peat, hypersaline mat, and freshwater, suggesting a considerable higher diversity and broader than expected habitat distribution for this phylum. Despite their small genomes (0.64-1.08 Mb), these archaea may contribute to carbon and nitrogen cycling by degrading multiple saccharides and proteins, and produce ATP via aerobic respiration and fermentation. Additionally, we identified several syntenic genes with homology to those involved in iron oxidation in six Parvarchaeota genomes, suggesting their potential role in iron cycling. However, both phyla lack biosynthetic pathways for amino acids and nucleotides, suggesting that they likely scavenge these biomolecules from the environment and/or other community members. Moreover, low-oxygen enrichments in laboratory confirmed our speculation that both phyla are microaerobic/anaerobic, based on several specific genes identified in them. Furthermore, phylogenetic analyses provide insights into the close evolutionary history of energy related functionalities between both phyla with Thermoplasmatales. These results expand our understanding of these elusive archaea by revealing their involvement in carbon, nitrogen, and iron cycling, and suggest their potential interactions with Thermoplasmatales on genomic scale.

Complementary Methodologies To Investigate Human Gut Microbiota in Host Health, Working towards Integrative Systems Biology.

In 1680, Antonie van Leeuwenhoek noted compositional differences in his oral and fecal microbiota, pioneering the study of the diversity of the human microbiome. From Leeuwenhoek's time to successful modern attempts at changing the gut microbial landscape to cure disease, there has been an exponential increase in the recognition of our resident microbes as part of ourselves. Thus, the human host and microbiome have evolved in parallel to configure a balanced system in which microbes survive in homeostasis with our innate and acquired immune systems, unless disease occurs. A growing number of studies have demonstrated a correlation between the presence/absence of microbial taxa and some of their functional molecules (i.e., genes, proteins, and metabolites) with health and disease states. Nevertheless, misleading experimental design on human subjects and the cost and lack of standardized animal models pose challenges to answering the question of whether changes in microbiome composition are cause or consequence of a certain biological state. In this review, we evaluate the state of the art of methodologies that enable the study of the gut microbiome, encouraging a change in broadly used analytic strategies by choosing effector molecules (proteins and metabolites) in combination with coding nucleic acids. We further explore microbial and effector microbial product imbalances that relate to disease and health.

Determinants and Prediction of Esterase Substrate Promiscuity Patterns.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

Bacterial, Archaeal, and Eukaryotic Diversity across Distinct Microhabitats in an Acid Mine Drainage.

Acid mine drainages are characterized by their low pH and the presence of dissolved toxic metallic species. Microorganisms survive in different microhabitats within the ecosystem, namely water, sediments, and biofilms. In this report, we surveyed the microbial diversity within all domains of life in the different microhabitats at Los Rueldos abandoned mercury underground mine (NW Spain), and predicted bacterial function based on community composition. Sediment samples contained higher proportions of soil bacteria (AD3, Acidobacteria), as well as Crenarchaeota and Methanomassiliicoccaceae archaea. Oxic and hypoxic biofilm samples were enriched in bacterial iron oxidizers from the genus Leptospirillum, order Acidithiobacillales, class Betaproteobacteria, and archaea from the class Thermoplasmata. Water samples were enriched in Cyanobacteria and Thermoplasmata archaea at a 3-98% of the sunlight influence, whilst Betaproteobacteria, Thermoplasmata archaea, and Micrarchaea dominated in acid water collected in total darkness. Stalactites hanging from the Fe-rich mine ceiling were dominated by the neutrophilic iron oxidizer Gallionella and other lineages that were absent in the rest of the microhabitats (e.g., Chlorobi, Chloroflexi). Eukaryotes were detected in biofilms and open-air water samples, and belonged mainly to clades SAR (Alveolata and Stramenopiles), and Opisthokonta (Fungi). Oxic and hypoxic biofilms displayed higher proportions of ciliates (Gonostomum, Oxytricha), whereas water samples were enriched in fungi (Paramicrosporidium and unknown microbial Helotiales). Predicted function through bacterial community composition suggested adaptive evolutive convergence of function in heterogeneous communities. Our study showcases a broad description of the microbial diversity across different microhabitats in the same environment and expands the knowledge on the diversity of microbial eukaryotes in AMD habitats.

Metabolic networks for nitrogen utilization in Prevotella ruminicola 23.

Nitrogen metabolism in gut systems remains poorly studied in spite of its importance for microbial growth and its implications for the metabolism of the host. Prevotella spp. are the most predominant bacteria detected in the rumen, but their presence has also been related to health and disease states in the human gut and oral cavity. To explore the metabolic networks for nitrogen assimilation in this bacterium, changes in gene expression profiles in response to variations in the available nitrogen source and to different concentrations of ammonium were analyzed by microarray and reverse transcription quantitative PCR, and linked with function by further proteomic analysis. The observed patterns of transcript abundances for genes involved in ammonium assimilation differed from the classical "enteric paradigm" for nitrogen utilization. Expression of genes encoding high substrate affinity nitrogen assimilation enzymes (GS-GOGAT system) was similar in growth-limiting and non-limiting nitrogen concentrations in P. ruminicola 23, whereas E. coli and Salmonella spp. responses to excess nitrogen involve only low substrate affinity enzymes. This versatile behavior might be a key feature for ecological success in habitats such as the rumen and human colon where nitrogen is rarely limiting for growth, and might be linked to previously reported Prevotella spp. population imbalances relative to other bacterial species in gut systems.

Identification and characterization of a 20ß-HSDH from the anaerobic gut bacterium Butyricicoccus desmolans ATCC 43058.

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20ß-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20ß-HSDH in both B. desmolans and C. cadaveris The 20ß-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20ß-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0-7.5 with (20ß-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with Km , 0.80 ± 0.06 µM; Vmax , 30.36 ± 1.97 µmol·min-1; Kcat , 607 ± 39 µmol·µM-1·min-1; Kcat /Km , 760 ± 7.67. Phylogenetic analysis of the 20ß-HSDH from B. desmolans suggested that the 20ß-HSDH is found in several Bifidobacterium spp, one of which was shown to express 20ß-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.

Exploring the human microbiome from multiple perspectives: factors altering its composition and function.

Our microbiota presents peculiarities and characteristics that may be altered by multiple factors. The degree and consequences of these alterations depend on the nature, strength and duration of the perturbations as well as the structure and stability of each microbiota. The aim of this review is to sketch a very broad picture of the factors commonly influencing different body sites, and which have been associated with alterations in the human microbiota in terms of composition and function. To do so, first, a graphical representation of bacterial, fungal and archaeal genera reveals possible associations among genera affected by different factors. Then, the revision of sequence-based predictions provides associations with functions that become part of the active metabolism. Finally, examination of microbial metabolite contents and fluxes reveals whether metabolic alterations are a reflection of the differences observed at the level of population structure, and in the last step, link microorganisms to functions under perturbations that differ in nature and aetiology. The utilisation of complementary technologies and methods, with a special focus on metabolomics research, is thoroughly discussed to obtain a global picture of microbiota composition and microbiome function and to convey the urgent need for the standardisation of protocols.

Antibiotic use and microbiome function.

Our microbiome should be understood as one of the most complex components of the human body. The use of ß-lactam antibiotics is one of the microbiome covariates that influence its composition. The extent to which our microbiota changes after an antibiotic intervention depends not only on the chemical nature of the antibiotic or cocktail of antibiotics used to treat specific infections, but also on the type of administration, duration and dose, as well as the level of resistance that each microbiota develops. We have begun to appreciate that not all bacteria within our microbiota are vulnerable or reactive to different antibiotic interventions, and that their influence on both microbial composition and metabolism may differ. Antibiotics are being used worldwide on a huge scale and the prescription of antibiotics is continuing to rise; however, their effects on our microbiota have been reported for only a limited number of them. This article presents a critical review of the antibiotics or antibiotic cocktails whose use in humans has been linked to changes in the composition of our microbial communities, with a particular focus on the gut, oral, respiratory, skin and vaginal microbiota, and on their molecular agents (genes, proteins and metabolites). We review the state of the art as of June 2016, and cover a total of circa 68 different antibiotics. The data herein are the first to compile information about the bacteria, fungi, archaea and viruses most influenced by the main antibiotic treatments prescribed nowadays.

Lignocellulose-derived thin stillage composition and efficient biological treatment with a high-rate hybrid anaerobic bioreactor system.

BACKGROUND: This study aims to chemically characterize thin stillage derived from lignocellulosic biomass distillation residues in terms of organic strength, nutrient, and mineral content. The feasibility of performing anaerobic digestion on these stillages at mesophilic (40 °C) and thermophilic (55 °C) temperatures to produce methane was demonstrated. The microbial communities involved were further characterized. RESULTS: Energy and sugar cane stillage have a high chemical oxygen demand (COD of 43 and 30 g/L, respectively) and low pH (pH 4.3). Furthermore, the acetate concentration in sugar cane stillage was high (45 mM) but was not detected in energy cane stillage. There was also a high amount of lactate in both types of stillage (35-37 mM). The amount of sugars was 200 times higher in energy cane stillage compared to sugar cane stillage. Although there was a high concentration of sulfate (18 and 23 mM in sugar and energy cane stillage, respectively), both thin stillages were efficiently digested anaerobically with high COD removal under mesophilic and thermophilic temperature conditions and with an organic loading rate of 15-21 g COD/L/d. The methane production rate was 0.2 L/g COD, with a methane percentage of 60 and 64, and 92 and 94 % soluble COD removed, respectively, by the mesophilic and thermophilic reactors. Although both treatment processes were equally efficient, there were different microbial communities involved possibly arising from the differences in the composition of energy cane and sugar cane stillage. There was more acetic acid in sugar cane stillage which may have promoted the occurrence of aceticlastic methanogens to perform a direct conversion of acetate to methane in reactors treating sugar cane stillage. CONCLUSIONS: Results showed that thin stillage contains easily degradable compounds suitable for anaerobic digestion and that hybrid reactors can efficiently convert thin stillage to methane under mesophilic and thermophilic conditions. Furthermore, we found that optimal conditions for biological treatment of thin stillage were similar for both mesophilic and thermophilic reactors. Bar-coded pyrosequencing of the 16S rRNA gene identified different microbial communities in mesophilic and thermophilic reactors and these differences in the microbial communities could be linked to the composition of the thin stillage.